4BNT162b2 mRNA COVID‐19 vaccine and semen: What do we know?

ABSTRACT Background The effects of an mRNA COVID‐19 vaccine on spermatozoa parameters are not known. The aim of this study was to evaluate the effect of the BNT162b2 mRNA COVID‐19 vaccine on human semen, comparing spermatozoa parameters before and after vaccine inoculation. Materials and methods In this single‐center prospective study, voluntary subjects who received mRNA vaccines from February to July 2021 were enrolled. The study population included male subjects aged between 18 and 45 years who completed the BNT162b2 mRNA COVID‐19 vaccine cycle. All subjects were evaluated before the first dose of vaccine (T0) and after 3 months (T1) with semen analysis and further analysis of seminal plasma, including colorimetric determination of reactive oxygen metabolites (d‐ROM test), electrolytes, and interleukin 6 (IL‐6) assessment by enzyme‐linked immunosorbent assay technology. Results The experimental sample included 47 subjects (age: 29.3 ± 6.0 years, range 24–32; body mass index: 23.15 ± 2.5 kg/m2, range 19.2–28.0). All the subjects reported no systemic side effects. No significant differences were observed in any spermatozoa parameter between T0 and T1. A subanalysis was performed in oligoazoospermic and asthenozoospermic subjects, confirming the same results. Electrolyte analysis also showed no significant differences before and after vaccine inoculation. Finally, no significant differences were observed in T0, compared to T1 for the d‐ROM test and IL‐6. Discussion and conclusion In this study, no significant differences in spermatozoa parameters before and after vaccine inoculations were found. Furthermore, oxidative stress analysis,, the activity of the cell membrane, and IL‐6, as a marker of inflammation, was not affected by the mRNA COVID‐19 vaccine. These results suggest that this vaccine is safe for male semen quality.

Several studies reported that SARS-CoV-2 infection could be associated with an impairment in testicular function. [4][5][6][7][8] However, few studies have been carried out to investigate the possible correlation between infection and spermatozoa parameter impairment. 5

Xu et al. reported structural changes in testicular cells (Leydig and
Sertoli cells as well as spermatogonia), which can affect the reproductive system; the analysis was conducted post-mortem in patients with SARS coronavirus infection. 9 In particular, vacuolation and cytoplasmic dilution of Sertoli cells, loss of integrity of the basement membrane, and seminiferous tubules and inflammatory cell infiltrate were observed.
This inflammatory infiltrate seems to be related to the presence of a high viral load for which protein S is responsible. 9 EMA published a data sheet that included potential adverse events of the COVID-19 vaccine. 3 Despite the few adverse events found in clinical trials, mainly characterized by short-term, mild-to-moderate pain at the injection site, fatigue, and headache, 10 very few data are available on the potential reproductive toxicity of the SARS-CoV-2 vaccine. In this regard, an animal study was performed in female mice; no significant alterations in reproductive function were observed following SARS-CoV-2 vaccination. 3,11 On the other hand, no studies were conducted to evaluate the reproductive function in males before the authorization of the vaccine.
In general, there are few studies concerning reproductive toxicity vaccine related; however, all studies are in agreement in stating that any alterations in spermatogenesis can occur mainly as a consequence of adverse reactions, such as an increase in body temperature above 38 • C, and represent a transitory effect. 12,13 On the other hand, Chaudhary et al. reported adverse reactions related to the use of vaccine excipients and/or adjuvants. 12 Previously, Xu et al. studied the reproductive toxicity of adjuvants such as silver nanoparticles 14 in rabies virus vaccines and highlighted that their small size can allow crossing through biological barriers such as the blood-testis barrier, inducing cytotoxicity (inflammatory state and a decrease in mitochondrial function with the production of reactive oxygen species [ROS] and an increase in cellular apoptosis and/or a downregulation of gene expression, which could induce cellular apoptosis). Moreover, cytotoxicity has been associated with electrolyte imbalance between the intracellular membrane and the extracellular membrane, which leads to an alteration of metabolic activities. 15,16 Furthermore, there is a lack of knowledge and understanding about newly developed mRNA-based vaccines induced in the popular media the fear of a potential association between the SARS-CoV-2 vaccine and male infertility. The confusion generated in public opinion has brought to light widespread concern about any short-term reactions but, above all, about the long-term side effects of vaccines.
To date, two studies have been conducted to investigate the relationship between spermatozoa parameters and the BNT162b2 mRNA Covid-19 vaccine. 17,18 In both studies, the authors found no significant abnormality in any spermatozoa parameter, but some drawbacks limit the significance of these analyses, of which the most relevant was that progressive motility and morphology were not considered 17 and that the analysis was performed approximativelyafter 1-2 months following their second dose. 18 The aim of this study was to evaluate the effect of the BNT162b2 mRNA Covid-19 vaccine on human semen, comparing spermatozoa parameters before and after vaccine inoculation. Furthermore, to exclude the negative effects of vaccines related to oxidative stress, cell membrane activity and inflammation, we also assessed reactive oxygen metabolites, electrolytes, and interleukin 6 (IL-6) in seminal plasma.

MATERIALS AND METHODS
In this single-center prospective study, voluntary subjects recruited

Semen analysis
Semen analysis was performed before the first dose of vaccine (T0) and after 3 months (T1), which corresponds to 70 days from the second dose ( Figure 1).
Seminal fluid of each participant was collected by masturbation after sexual abstinence between 2 and 7 days. All samples were allowed to liquefy at 37 • C for 60 mins and were then assessed according to World Health Organization (WHO). 19 Macroscopic examination (volume, appearance, pH, liquefaction, viscosity) and microscopic evaluation were carried out; in particular, morphology assessment was carried out after smear staining with the Bryan-Leishman method. 20,21 The following seminal parameters were evaluated: sperm concentration (10 6 /ml), total spermatozoa number (n × 106/ejaculate), total motility and progressive motility (%), morphology (% abnormal forms), and round cells (n x10 6 /ml

Colorimetric d-ROMs
In

Electrolytes
The electrolytes were assessed by automated instrumentation. Architect cSystems A total of 300 μl of each sample was used for the assay.

Statistical analysis
All statistical analyses were performed using R version 4.0.3 (2020-10-

RESULTS
A total of 52 subjects were enrolled. Of them, five subjects were excluded (two were azoospermic; three subjects took drugs after the first vaccine dose).  Table 2).
Finally, no significant correlations were observed between all spermatozoa parameters and electrolytes as well as d-ROM test and IL-6, with the exception of a weak inverse correlation between T0 sodium and T1 IL-6 (r = −0.2).

DISCUSSION
Testicular damage and subsequent infertility have been hypothesized to be a consequence of COVID-19 infection. 4 Several studies have shown that SARS-CoV-2 testis localization can increase the forthcoming risk of spermatozoa production impairment and hypogonadism. 6,7 Similarly, low testosterone in the acute phase of the disease can increase the risk of worse outcomes. however, false positive results could be due to contamination with respiratory droplets in the semen containers. 26 As a consequence, couples delayed planning their pregnancies for fear of infection. 27,28 Furthermore According to these data, in the present study, we found no significant differences in sperm concentration, motility, and morphology when comparing values before and after vaccine inoculations. We also analyzed the effects of the vaccine in subjects with oligo/asthenozoospermia, and we observed the same results. Moreover, an improving trend in all spermatozoa parameters could be noted in this subgroup, particularly in motility. However, according to Gonzalez et al., 17 this improvement can be associated with the normal individual variation in spermatozoa parameters.
Furthermore, we also evaluated reactive oxygen metabolites, electrolytes, and IL-6 in seminal plasma. Interestingly, we found no differences in the d-ROM test, expression of oxidative stressand free radical-derived compounds. Recent evidence showed that oxidative stress could increase the affinity of SARS-CoV and SARS-CoV-2 protein "S" for the angiotensin-converting enzyme 2 receptor; this mechanism, therefore, could increase the severity of COVID-19 infection. 30 On the other hand, it is well known that oxidative stress could play an independent role in the etiology semen impairment by hindering the capacitation process and damaging spermatozoa membrane and DNA. 31 In this regard, approximately 30%-80% of infertile men showed higher seminal ROS levels. 31 The analysis of the electrolytes showed good cell membrane activity, which was stable after vaccine inoculation. In fact, an electrolytic balance must be present between the intracellular membrane (chlorine, calcium, magnesium, potassium) and extracellular space (sodium) for the cell to carry out its normal functions. 15 Defective expression of each electrolyte has been described in several studies on the basis of spermatogenic dysfunctions; in this regard, electrolyte analyses of human seminal fluid have been previously performed. 16,32,33 Specifically, Nag et al. observed a significantly lower Na concentration in asthenozoospermic subjects than in normospermic subjects. 32 Furthermore, Bondani et al. showed that K levels were higher in oligoasthenospermic subjects than in normospermic subjects. 16 Considering IL-6 dosage, a marker of inflammation, the analysis revealed no significant differences before and after vaccination. It is known that the IL-6 concentration in seminal plasma is higher in infertile men and correlates with reduced motility. 34 Furthermore, high IL-6 levels in blood seem to be associated with adverse clinical outcomes in patients with COVID-19. 35 Finally, we could speculate that the weak inverse correlation between T0 sodium and T1 IL-6 indicates an inverse correlation between better membrane activity 32 and lower inflammation.
The main limitations of this study are the small number of oligozoospermic individuals, the group of subjects likely most susceptible to worsening semen parameters, and that the study group came from a specific population and were not random people.

CONCLUSION
In conclusion, no significant differences were observed between semen parameters, oxidative stress analysis, or IL-6 as a marker of inflammation and electrolyte function before and after the BNT162b2 mRNA COVID-19 vaccine. Our results suggest that this kind of COVID-19 vaccine is safe for male semen quality.